Jnk3 modulators and methods of use

ABSTRACT

The c-Jun NH 2 -terminal kinase (JNK) group of MAP kinases are activated by exposure of cells to environmental stress. The role of JNK in the brain was examined by targeted disruption of the gene that encodes the neuronal isoform JNK3. It was found that JNK3 is required for the normal response to seizure activity. Methods of screening for molecules and compounds that decrease JNK3 expression or activity are described. Such molecules or compounds are useful for treating disorders involving excitotoxicity such as seizure disorders, Alzheimer&#39;s disease, Huntington disease, Parkinson&#39;s disease, and ischaemia.

This application claims priority from provisional application Ser. No.60/060,995, filed Oct. 3, 1997.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made, in part, with support from the NationalInstitutes of Health. The government may have certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to screening assays for the detection ofinhibitors of protein kinase expression or activity.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, is a prominent feature of thenervous system during normal development and in adult brain exposed toenvironmental stress (Kuida et al., Nature, 384:368-372, 1996; Ratan etal., Neurochem., 62:376-379, 1994; Raff et al., Science, 262:695-700,1993). Stress-induced apoptosis has been implicated in a variety ofneurological diseases (Thompson, Science, 267:1456-1462, 1995) andrequires de novo protein and RNA synthesis (Martin et al., J. Cell.Biol., 106:829-844, 1988; Oppenheim et al., Dev. Biol., 138:104-113,1990). Increased expression of c-Jun protein is associated with neuronaldamage following global ischemia (Neumann-Haefelin et al., Cerebral FlowMetab, 14:206-216, 1994) or transection of nerve axons in vivo(Neumann-Haefelin, supra). Increased expression and phosphorylation ofc-Jun have been observed in vitro prior to the apoptotic death ofsympathetic neurons deprived of nerve growth factor (NGF) (Ham et al.,Neuron, 14:927-939, 1995). Moreover, expression of a dominant negativemutant c-Jun, or treatment with c-Jun antibody protects NGF-deprivedsympathetic neurons from apoptosis (Ham et al., supra; Estus et al., J.Cell. Biol., 127:1717-1727, 1994). However, the requirement of c-Jun forstress-induced neuronal apoptosis has not been tested in vivo sincec-Jun deficient mice die during mid-gestation (Hilberg et al., Nature,365:179-181, 1993).

Protein phosphorylation is one important mechanism involved in theactivation of c-Jun in response to environmental stress signals(Whitmarsh et al., J. Mol. Med., 74:589-607, 1996). c-Jun N-terminalkinase (JNK, also known as SAPK) is a serine/threonine protein kinasethat phosphorylates two residues (Ser-63 and Ser-73) on the NH₂-terminalactivation domain of c-Jun (Whitmarsh et al., supra; Dèrijard et al.,Cell, 76:1025-1037, 1994; Kyriakis et al., Nature, 369:156-160, 1994).Map kinase kinase (MKK) 4 (also known as SEK1) is a direct activator ofJNK in response to environmental stresses and mitogenic factors(Whitmarsh et al, supra; Dèrijard et al, supra; Nishina et al., Nature,385:350-353, 1997; Yang et al., Proc. Nat. Acad. Sci. USA, 94:3004-3009,1997; Sanchez et al., Nature, 372:794-798, 1994). JNK alsophosphorylates ATF2 and other Jun-family proteins which function ascomponents of the AP-1 transcription factor complex (Whitmarsh et al.,supra; Gupta et al., Science, 267:389-393, 1995; Gupta et al., EMBO J.,15:2760-2770, 1996).

The phosphorylation of these transcription factors by JNK leads toincreased AP-1 transcriptional activity (Whitmarsh et al., supra).Conversely, the induction of AP-1 transcriptional activity isselectively blocked in cells lacking MKK4 (Yang et al., supra).

JNK has been implicated in the apoptosis of NGF-differentiated PC12pheochromocytoma cells (Xia et al., Science, 270:1326-1311, 1995), onemodel system of neuronal cell death in vivo (Batistatou et al., J. Cell.Biol., 122:523-532, 1993). When differentiated PC12 cells are deprivedof nerve growth factor (NGF), JNK activation is observed prior toapoptotic death (Xia et al., supra). Transfection studies usingconstitutively activated and dominant negative mutant components of theJNK signaling pathway established that JNK is involved in NGFwithdrawal-induced apoptosis of PC12 cells (Xia et al., supra).

Ten JNK isoforms, resulting from alternative splicing of three differentgenes have been identified (Dèrijard et al., supra; Kyriakis et al.,supra; Gupta et al., supra; Martin et al., Brain Res. Mol. Brain. Res.,35:47-57, 1996). Although the JNK1 and JNK2 isoforms are widelyexpressed in murine tissues, including the brain, the JNK3 isoforms arepredominantly expressed in the brain and, to a lesser extent, in theheart and testis.

SUMMARY OF THE INVENTION

The invention is based on the discovery that mice lacking the JNK3 gene(JNK3(−/−)) develop normally and are resistant to excitotoxic damage,and that JNK3 plays a role in stress-induced seizure activity, AP-1transcriptional activation, and kainate-induced apoptosis of hippocampalneurons. Thus, JNK3 is a mediator of kainate/glutamate excitotoxicityand a target for limiting or preventing excitotoxic damage.

The invention also features a method of identifying a candidate compoundthat modulates JNK3 expression. The method includes the steps ofincubating a cell that can express a JNK3 protein with a compound underconditions and for a time sufficient for the cell to express the JNK3protein when the candidate compound is not present. The expression ofJNK3 is then measured in the cell in the presence of the compound. Theexpression of JNK3 is also measured in a control cell under the sameconditions and for the same time. The amount of JNK3 expression in thecell incubated in the presence of the compound and in the control cellis compared. A difference in JNK3 expression indicates that the compoundmodulates JNK3 expression. In an embodiment of this method, the compounddecreases JNK3 expression.

In another embodiment, the invention features a method of identifying acandidate compound that modulates JNK3 activity. The method includes thesteps of incubating a cell that has JNK3 activity with a compound underconditions and for a time sufficient for the cell to express JNK3activity when the candidate compound is not present. The activity ofJNK3 is then measured in the cell in the presence of the compound. Theactivity of JNK3 is also measured in a control cell under the sameconditions and for the same time. The amount of JNK3 activity in thecell incubated in the presence of the compound and in the control cellis compared. A difference in JNK3 activity indicates that the compoundmodulates JNK3 activity. In an embodiment of this method, the compounddecreases JNK3 activity.

The invention also includes a method of identifying a compound thatmodulates the binding of a JNK3 polypeptide to a substrate. The methodinvolves comparing the amount of a JNK3 polypeptide bound to a substratein the presence and absence of a selected compound. A difference in theamount of binding of a JNK3 polypeptide to the substrate indicates thatthe selected compound modulates the binding of a JNK3 polypeptide. In anembodiment of this method, the binding of a JNK3 polypeptide to asubstrate is decreased.

Another feature of the invention is a method for generating a totipotentmouse cell comprising at least one inactivated JNK3 gene. The methodincludes: a) providing a plurality of totipotent mouse cells; b)introducing into the cells a DNA construct that includes a mouse JNK3gene disrupted by the insertion of a sequence into the gene, thus thedisruption prevents expression of functional JNK3; c) incubating thecells so that homologous recombination occurs between the chromosomalsequence encoding JNK3 and the introduced DNA construct; and d)identifying a totipotent mouse cell that has at least one inactivatedJNK gene.

Also featured in the invention is a method for generating a mousehomozygous for an inactivated JNK3 gene. The method includes the stepsof: a) providing a totipotent mouse cell that contains at least oneinactivated JNK3 gene; b) inserting the cell into a mouse embryo andimplanting the embryo into a female mouse; c) permitting the embryo todevelop into a neonatal mouse; d) permitting the neonatal mouse to reachsexual maturity; e) mating two of the sexually mature mice to obtain amouse homozygous for the inactivated JNK3 gene. Such a mouse (homozygousJNK3(−/−)) is resistant to excitotoxic damage.

The invention also features methods of treating a patient having or atrisk for a disorder of the nervous system involving excitotoxicity. Themethods include administering to the patient a therapeutically effectiveamount of a compound that inhibits JNK3 expression, or a therapeuticallyeffective amount of a compound that inhibits JNK3 activity. An antisensenucleic acid molecule or ribozyme can be used as the inhibitorycompound. Disorders that can be treated by these methods includedementias including Alzheimer's disease, neurodegenerative diseases suchas Huntington disease, cerebrovascular disorders such as ischemia,amyotrophic lateral sclerosis, trauma including that caused by heat orcold, motor neuron disease, Parkinson's disease, or seizure disordersincluding epilepsy. Neuroendocrine disorders such as those that affectpituitary glands, adrenal glands, testis, or pancreas (e.g., β-cells)can be treated with JNK3 modulators.

The invention also includes a transgenic non-human mammal having atransgene disrupting expression of a JNK3 gene, the transgene beingchromosomally integrated into germ cells of the mammal. In an embodimentof the invention, the mammal is a mouse. The germ cells of the mammalcan be homozygous for the transgene and the disruption of JNK3 geneexpression can be the result of a null mutation. Another embodiment ofthe invention includes a cell line descended from a cell of the mammalhaving the transgene disrupting expression of a JNK3 gene.

A DNA construct comprising a disrupted mouse JNK3 gene is also featuredin the invention. The disruption is by insertion of a sequence into thegene such that the disruption prevents or modifies the expression offunctional JNK3.

Unless otherwise specified, “JNK3” can refer both to nucleic acids andpolypeptides, such as the sequences shown in FIGS. 1A-5B (SEQ IDNOS:1-12; see also, GenBank accession number: U34819 which correspondsto SEQ ID NO:1 and SEQ ID NO:2; U34820 which corresponds to SEQ ID NO:4and SEQ ID NO:5; U07620 which corresponds to SEQ ID NO:7 and SEQ IDNO:8; L27128 which corresponds to SEQ ID NO:9 and SEQ ID NO:10; andL35236 which corresponds to SEQ ID NO:11 and SEQ ID NO:12). SEQ ID NO:3and SEQ ID NO:6 represent deduced nucleotide sequences based on thepresumed overlap between the sequences represented by SEQ ID NOS:1 and 4with the sequence represented by SEQ ID NO:7. JNK3 also refers topolypeptides that are at least 85% identical to the amino acid sequenceslisted above, and to the nucleic acids encoding those polypeptides.Examples of these sequences and methods of isolating them are found inGupta et al., supra, 1996; Kyriakis et al., supra; Martin et al., BrainRes. Mol. Brain. Res., 35:45-57, 1996; and Mohit et al., Neuron,14:67-78, 1995.

A “control” cell is a cell that is generally the same, e.g.,genotypically and phenotypically, as the cell to which it is beingcompared (e.g., the cells can be sister cells), but which is not exposedto a test compound.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the nucleic acid sequence ofGenBank Accession No. U34819 (SEQ ID NO:1).

FIG. 1B is a schematic representation of the amino acid sequence ofGenBank Accession No. U34819 (SEQ ID NO:2).

FIG. 1C is a schematic representation of the nucleic acid sequence ofSEQ ID NO:3.

FIG. 2A-B is a schematic representation of the nucleic acid sequence ofGenBank Accession No. U34820 (SEQ ID NO:4).

FIG. 2C is a schematic representation of the amino acid sequence ofGenBank Accession No. U34820 (SEQ ID NO:5).

FIG. 2D is a schematic representation of the nucleic acid sequence ofSEQ ID NO:6.

FIG. 3A-B is a schematic representation of the nucleic acid sequence ofGenBank Accession No. U07620 (SEQ ID NO:7).

FIG. 3C is a schematic representation of the amino acid sequence ofGenBank Accession No. U07620 (SEQ ID NO:8).

FIG. 4A is a schematic representation of the nucleic acid sequence ofGenBank Accession No. L27128 (SEQ ID NO:9).

FIG. 4B is a schematic representation of the amino acid sequence ofGenBank Accession No. L27128 (SEQ ID NO:10).

FIG. 5A is a schematic representation of the nucleic acid sequence ofGenBank Accession No. L35236 (SEQ ID NO:11)

FIG. 5B is a schematic representation of the amino acid sequence ofGenBank Accession No. L35236 (SEQ ID NO:12).

FIG. 6 is a diagram of the wild type JNK3 gene locus, targeting vector,and the mutated or disrupted JNK3 gene locus.

FIG. 7 is a bar graph showing the temporal responses of wild type andJNK3(−/−) mice to kainic acid (KA) injection.

FIG. 8 is a bar graph showing the temporal responses of wild type andJNK3(−/−) mice to pentetrazole (PTZ) injection.

FIG. 9A is a schematic representation of the nucleic acid sequence ofmurine c-Jun (GenBank Accession No. X12740; SEQ ID NO:13).

FIG. 9B is a schematic representation of the amino acid sequence ofmurine c-Jun (GenBank Accession No. X12740; SEQ ID NO:14).

FIG. 10A-B is a schematic representation of the nucleic acid sequence ofmurine c-Fos (GenBank Accession No. V00727; SEQ ID NO:15).

FIG. 10C is a schematic representation of the amino acid sequence ofmurine c-Fos (GenBank Accession No. V00727; SEQ ID NO:16)

FIG. 11 is a bar graph showing the level of KA-induced AP-1 activity atvarious times after KA induction as reflected by luciferase activity inJNK3(−/−) mice crossed with transgenic AP-1 luciferase mice.

FIG. 12 is a bar graph showing the level of KA-induced AP-1 activity asreflected by the relative level of luciferase activity in hippocampus(HP) and cerebellum (CB) of JNK3(−/−) mice and wild type (+/+) mice.

FIG. 13 is a diagram of the proposed chain of molecular events caused byKA leading to neuronal apoptosis.

FIG. 14 is a diagram of the trisynaptic connection within thehippocampal formation.

DETAILED DESCRIPTION

JNK protein kinase phosphorylates c-Jun and subsequently increases AP-1transcriptional activity in response to a specific group of stresssignals (Whitmarsh et al, supra; Yang et al., supra). Theneural-specific expression of JNK3 may render neurons particularlysusceptible to physiological stress. In the experiments describedherein, a remarkable resistance to kainic acid (KA)-induced seizures andapoptosis has been observed in JNK3-deficient mice. The resistance to KAneurotoxicity may be due to the elimination of a specificstress-response pathway mediated by the JNK3 isoform of JNK proteinkinase. First, the administration of KA caused the phosphorylation ofthe NH₂-terminal activation domain of c-Jun and markedly increased AP-1transcriptional activity in wild-type, but not in JNK3-deficient mice.Second, there was prolonged expression of phosphorylated c-Jun withinthe most vulnerable area of the hippocampus, further indicating that JNKactivity may lead to neuronal apoptosis.

The findings reported herein are consistent with the dependence of KAneurotoxicity on excitatory circuitry (Nadler et al., Brain Res.195:47-56, 1980). Since JNK3 is widely expressed in the nervous systemand its activity is increased by many different stress signals (Gupta etal., supra), JNK3 may be involved in stress-induced apoptosis caused bya wide range of environmental insults.

The identification of JNK3 as a critical mediator of KA-inducedexcitatory neurotoxicity has clinical implications. The amino acidsequence of mouse, rat and human JNK3 is highly conserved (Kyriakis etal., supra; Gupta et al., supra; Martin et al., supra; Mohit et al.,Neuron. 14:67-78, 1995). Moreover, the expression of the human JNK3 geneis also restricted to the nervous system and neuroendocrine system, iswidely expressed in many brain subregions (Gupta et al., supra; Mohit etal., supra). It is therefore likely that the human and rodent JNK3protein kinases have related or identical physiological functions.Neurotoxicity of the excitatory amino acids has been implicated in manyneurological disorders ranging from acute ischemia to chronicneurodegenerative diseases (Choi, Neuron, 1:623-634, 1988; Lipton etal., N. Engl. J. Med. 330:613-622, 1994; Rothman et al., Annu. Neurol.19:105-111, 1986). Previous therapeutic strategies have been focused onthe prevention of calcium influx through cell surface channels, such asthe NMDA-type glutamate receptor. To date, these approaches have onlymet with mixed results (Lipton et al., supra). JNK3 is therefore atarget for therapeutic interventions when excitatory neurotoxicityinvolves JNK3-mediated apoptosis.

In the experiments described infra, homologous recombination was used togenerate JNK3-deficient mice, and their responses to noxious stimuliwere examined. KA, a potent excitotoxic chemical, elicits limbicseizures and neuronal cell death. The neurotoxicity of KA derives fromthe direct stimulation of the glutamate receptor at postsynaptic sites,and an indirect increase in the release of excitatory amino acids frompresynaptic sites. It is well-documented that systemic application of KAinduces the expression of various cellular immediate early genes(cIEGs), including c-Jun and c-Fos. Thus, the application of KA triggersa stress-response pathway in the brain in vivo. The experiments detailedinfra demonstrate that KA induces phosphorylation of c-Jun and anincrease in AP-1 transcriptional activity in the brain of wild-typemice. However, these effects of KA are markedly suppressed in the brainsof JNK3-deficient mice. Moreover, JNK3-deficient mice exhibit aremarkable resistance to KA-induced seizures and apoptosis ofhippocampal neurons. These normal mice treated with KA represent auseful model of human disorders of the nervous system involvingexcitotoxicity.

Based on these experimental results, JNK3 was found to be an exceptionaltarget for limiting excitotoxic damage. In particular, JNK3 is a targetin screening protocols including protocols to screen for molecules thatregulate JNK3 gene expression, JNK3 binding to its substrates, and JNK3activity, as described below. The molecules found in these screens thateffectively decrease JNK3 expression or activity are candidate drugs tobe used to treat disorders of the nervous system involvingexcitotoxicity, including seizure disorders such as epilepsy,cerebrovascular disorders including ischemia, metabolic imbalance (e.g.,hypoglycemia), injury due to extreme heat or cold, trauma (e.g.,irradiation, spinal cord injury, pressure, and ionic imbalance),dementias such as Alzheimer's disease, Parkinson's disease, andneurodegenerative disorders (e.g., Huntington disease), and motoneurondisease (including amyotrophic lateral sclerosis) (Thompson, Science,267:1456-1462, 1995; Coyle et al., Science, 262:689-695, 1993).

Methods of Screening for Molecules that Inhibit JNK3 Activity

The following assays and screens can be used to identify compounds thatare effective inhibitors of JNK3 activity. The assays and screens can bedone by physical selection of molecules from libraries, and computercomparisons of digital models of compounds in molecular libraries and adigital model of the JNK3 active site. The inhibitors identified in theassays and screens may act by, but are not limited to, binding to JNK3(e.g., from mouse or human), binding to intracellular proteins that bindto JNK3, compounds that interfere with the interaction between JNK3 andits substrates, compounds that modulate the activity of a JNK3 gene, orcompounds that modulate the expression of a JNK3 gene or a JNK3 protein.

Assays can also be used to identify molecules that bind to JNK3regulatory sequences (e.g., promoter sequences), thus modulating geneexpression. See, e.g., Platt, J. Biol. Chem., 269:28558-28562, 1994.

The compounds that can be screened by the methods described hereininclude, but are not limited to, peptides and other organic compounds(e.g., peptidomimetics) that bind to a JNK3 protein or inhibit itsactivity in any way. Such compounds may include, but are not limited to,peptides; for example, soluble peptides, including but not limited tomembers of random peptide libraries (see, e.g., Lam et al., Nature354:82-84, 1991; Houghten et al., Nature 354:84-86, 1991), andcombinatorial chemistry-derived molecular libraries made of D- and/orL-amino acids, phosphopeptides (including, but not limited to, membersof random or partially degenerate, directed phosphopeptide libraries;see, e.g., Songyang et al., Cell 72:767-778, 1993), and small organic orinorganic molecules.

Compounds and molecules are screened to identify those that affectexpression of a JNK3 gene or some other gene involved in regulating theexpression of JNK3 (e.g., by interacting with the regulatory region ortranscription factors of a gene). Compounds are also screened toidentify those that affect the activity of such proteins (e.g., byinhibiting JNK3 activity) or the activity of a molecule involved in theregulation of JNK3.

Computer modeling or searching technologies are used to identifycompounds, or identify modified compounds that modulate or arecandidates to modulate the expression or activity of a JNK3 protein. Forexample, compounds likely to interact with the active site of the JWK3protein are identified. The active site of JNK3 can be identified usingmethods known in the art including, for example, analysis of the aminoacid sequence of a molecule, and from a study of complexes formed byJNK3 with a native ligand (e.g., ATF2 or c-Jun). Chemical or X-raycrystallographic methods can be used to identify the active site of JNK3by the location of a bound ligand such as c-Jun or ATF2.

The three-dimensional structure of the active site can be determined.This can be done using known methods, including X-ray crystallography,which can be used to determine a complete molecular structure. Solid orliquid phase NMR can be used to determine certain intra-moleculardistances. Other methods of structural analysis can be used to determinepartial or complete geometrical structures. Geometric structure can bedetermined with a JNK3 protein bound to a natural (e.g., c-Jun or ATF2)or artificial ligand which may provide a more accurate active sitestructure determination.

Computer-based numerical modeling can be used to complete an incompleteor insufficiently accurate structure. Modeling methods that can be usedare, for example, parameterized models specific to particularbiopolymers such as proteins or nucleic acids, molecular dynamics modelsbased on computing molecular motions, statistical mechanics models basedon thermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups are necessary, and can be selected fromforce fields known in physical chemistry. Information on incomplete orless accurate structures determined as above can be incorporated asconstraints on the structures computed by these modeling methods.

Having determined the structure of the active site of a JNK3 protein,either experimentally, by modeling, or by a combination of methods,candidate modulating compounds can be identified by searching databasescontaining compounds along with information on their molecularstructure. The compounds identified in such a search are those that havestructures that match the active site structure, fit into the activesite, or interact with groups defining the active site. The compoundsidentified by the search are potential JNK3 modulating compounds.

These methods may also be used to identify improved modulating compoundsfrom an already known modulating compound or ligand. The structure ofthe known compound is modified and effects are determined usingexperimental and computer modeling methods as described herein. Thealtered structure is compared to the active site structure of a JNK3protein to determine or predict how a particular modification to theligand or modulating compound will affect its interaction with thatprotein. Systematic variations in composition, such as by varying sidegroups, can be evaluated to obtain modified modulating compounds orligands of preferred specificity or activity.

Given the teachings herein, additional experimental and computermodeling methods useful to identify modulating compounds based onidentification of the active sites of a JNK3 protein and relatedtransduction and transcription factors can be developed by those skilledin the art.

Examples of molecular modeling systems are the QUANTA programs, e.g.,CHARMm, MCSS/HOOK, and X-LIGAND, (Molecular Simulations, Inc., SanDiego, Calif.). QUANTA provides a modeling environment for twodimensional and three dimensional modeling, simulation, and analysis ofmacromolecules and small organics. Specifically, CHARMm analyzes energyminimization and molecular dynamics functions. MCSS/HOOK characterizesthe ability of an active site to bind a ligand using energeticscalculated via CHARMm. X-LIGAND fits ligand molecules to electrondensity patterns of protein-ligand complexes. The program also allowsinteractive construction, modification, visualization, and analysis ofthe behavior of molecules with each other.

Articles reviewing computer modeling of compounds interacting withspecific proteins can provide additional guidance. For example, see,Rotivinen et al., Acta Pharmaceutical Fennica 97:159-166, 1988; Ripka,New Scientist 54-57 (Jun. 16, 1988); McKinaly and Rossmann, Ann. Rev.Pharmacol. Toxicol. 29:111-122, 1989; Perry and Davies, OSAR:Quantitative Structure-Activity Relationships in Drug Design pp. 189-193(Alan R. Liss, Inc., 1989); Lewis and Dean, Proc. R. Soc. Lond.236:125-140, 141-162, 1989; and, regarding a model receptor for nucleicacid components, see Askew et al., Am. J. Chem. Soc. 111:1082-1090.Computer programs designed to screen and depict chemicals are availablefrom companies such as MSI (supra), Allelix, Inc. (Mississauga, Ontario,Canada), and Hypercube, Inc. (Gainesville, Fla.). These applications arelargely designed for drugs specific to particular proteins; however,they may be adapted to the design of drugs specific to identifiedregions of DNA or RNA. Commercial sources of chemical libraries can beused as sources of candidate compounds. Such chemical libraries can beobtained from, for example, ArQule, Inc. (Medford, Mass.).

In addition to designing and generating compounds that alter binding, asdescribed above, libraries of known compounds, including naturalproducts, synthetic chemicals, and biologically active materialsincluding peptides, can be screened for compounds that are inhibitors oractivators.

Compounds identified by methods described above may be useful, forexample, for elaborating the biological function of JNK3 gene productsand in treatment of disorders in which JNK3 activity is deleterious.Assays for testing the effectiveness of compounds such as thosedescribed herein are further described below.

In Vitro Screening Assays for Compounds that Bind to JNK3 Proteins andGenes

In vitro systems can be used to identify compounds that can interact(e.g., bind) to JNK3 proteins or genes encoding those proteins. Suchcompounds may be useful, for example, for modulating the activity ofJNK3 polypeptides or nucleic acids, elaborating their biochemistry, ortreating disorders caused or exacerbated by JNK3 expression. Thesecompounds may themselves disrupt normal function or can be used inscreens for compounds that disrupt normal function.

Assays to identify compounds that bind to JNK3 proteins involvepreparation of a reaction mixture of the protein and the test compoundunder conditions sufficient to allow the two components to interact andbind, thus forming a complex that can be detected and/or isolated.

Screening assays for molecules that can bind to a JNK3 protein ornucleic acid can be performed using a number of methods. For example, aJNK3 protein, peptide, or fusion protein can be immobilized onto a solidphase, reacted with the test compound, and complexes detected by director indirect labeling of the test compound. Alternatively, the testcompound can be immobilized, reacted with JNK3 polypeptide, and anycomplexes detected. Microtiter plates can be used as the solid phase andthe immobilized component anchored by covalent or noncovalentinteractions. Non-covalent attachment may be achieved by coating thesolid phase with a solution containing the molecule, and drying.Alternatively, an antibody specific for JNK3 is used to anchor themolecule to the solid surface. Such surfaces may be prepared in advanceof use, and stored. JNK3 antibodies can be produced using conventionalmethods such as those described in Coligan et al. (Current Protocols inImmunology, John Wiley & Sons, Inc., 1994, see Volume 1, chapter 2).

In the assay, the non-immobilized component is added to the coatedsurface containing the immobilized component under conditions thatpermit interaction and binding between the two components. The unreactedcomponents are then removed (e.g., by washing) under conditions suchthat any complexes formed will remain immobilized on the solid phase.The detection of the complexes can be accomplished by a number ofmethods known to those in the art. For example, the nonimmobilizedcomponent of the assay may be prelabeled with a radioactive or enzymaticlabel and detected using appropriate means. If the non-immobilizedentity was not prelabeled, an indirect method is used. For example, ifthe non-immobilized entity is a JNK3 polypeptide, an antibody againstJNK3 is used to detect the bound molecule, and a secondary, labeledantibody is used to detect the entire complex.

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected (e.g., using an immobilized antibody specific for a JNK3protein).

Cell-based assays can be used to identify compounds that interact withJNK3 proteins. Cell lines that naturally express such proteins or havebeen genetically engineered to express such proteins (e.g., bytransfection or transduction with JNK3 DNA) can be used. For example,test compounds can be administered to cell cultures and thephosphorylation of ATF2 or c-Jun measured as described infra. A decreasein the amount of phosphorylation of a JNK3 substrate in the presence ofthe test compound compared to controls that do not contain the testcompound indicates that the test compound is an inhibitor of JNK3activity.

Inhibitors of JNK3 expression that act on the JNK3 promoter can beidentified using a chimeric gene in which genomic sequences includingthe JNK3 promoter are fused to a reporter, for example fireflyluciferase. Cultured cells (including neurons) transformed with this DNAare screened for the expression of luciferase activity. Compounds thatinhibit luciferase activity in this high throughput assay can beconfirmed by direct measurement of the endogenous JNK3 protein (byWestern blotting) and JNK3 mRNA (by Northern blotting) using methodsknown in the art (for example, see Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, 1994).

Candidate inhibitory compounds can be tested further in cell or tissuecultures as well as animal models. For example, cells expressing JNK3are incubated with a test compound. Lysates are prepared from treatedand untreated cells and Western blotted according to known methods. Theblots are probed with antibodies specific for JNK3. A decrease in theamount of JNK3 expression in cultures treated with the test compoundcompared to untreated controls indicates that the test compound is acandidate for a drug to treat disorders associated with JNK3 expression.

Assays for Compounds that Interfere with JNK3/JNK3 SubstrateInteractions

Molecules that disrupt the interaction between JNK3 and its substratescan be identified using assays that detect protein-protein interactions.For example, the yeast two-hybrid method detects protein interactions invivo. However, an in vitro assay is preferable because candidatemolecules may not be permeable to the yeast cell wall. An example of anin vitro assay for such test molecules that disrupt the interactionbetween JNKC3 and a substrate includes the use of immobilized JNK3 orimmobilized substrate (e.g., c-Jun) and incubation of the immobilizedcomponent with cell lysates or purified proteins in the presence andabsence of a test molecule. In general, the test molecule is tested overa range of a 100 fold molar excess over the most abundant component(e.g., the component immobilized or in solution). If the test moleculeis predicted to interact with the immobilized component of the assay,then it can be pre-incubated with that component before adding the celllysate or purified protein. After washing away unbound material, thebound proteins are detected with antibodies (e.g., ELISA or Westernblot) or through the use of labeled proteins (e.g. radioactive orfluorescent) using methods known in the art. Test molecules thatdecrease the amount of substrate bound to JNK3 are thus identified asmolecules that interfere with JNK3/JNK3 substrate interactions.

Assays for Compounds that Ameliorate the Effects of JNK3 In Vivo

Compounds identified as above, or other candidate compounds that inhibitJNK3 activity in vitro may be useful for treating disorders involvingJNK3 activity. These compounds can be tested in in vivo assays, forexample, in animal models of disorders involving JNK3 activity. Forexample, transgenic mouse models of ALS have been described (Bruijn andCleveland, Neuropathol. Appl. Neurobiol. 22:373-387, 1996; Dal Canto andGurney, Brain Res. 676: 25-40, 1995; Cleveland et al., Neurology 47:Suppl 2, S54-61) as have transgenic models of Alzheimer's disease suchas the PDAPP mouse and others (for examples, see Loring et al.,Neurobiol. Aging 17:173-182, 1996). MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced dopaminergicneurotoxicity has been used as a model for Parkinson's disease inrodents and nonhuman primates (for example, Przedborski et al., Proc.Nat'l. Acad. Sci. USA 93:4565-4571, 1996).

Test compounds predicted to inhibit JNK3 activity are administered toanimals, e.g., as described above, that serve as models for the variousdisease paradigms. Treated animals are then assayed for inhibition ofJNK3 activity. Such assays may be indirect or inferential, for example,improved health or survival of the animal indicates the efficacy of atest compound. Assays can also be direct, for example, a decrease inJNK3 or c-Jun expression can be measured by Northern analysis of neuraltissue removed from an animal treated with a test compound. A decreasein the amount of JNK3 mRNA present in the sample from treated animalscompared to untreated controls indicates that the test compound isinhibiting JNK3 expression. A decrease in the amount of c-Jun indicatesthat the test compound is inhibiting JNK3 expression or activity.

Antisense Constructs and Therapies

Treatment regimes based on an “antisense” approach involve the design ofoligonucleotides (either DNA or RNA) that are complementary to JNK3mRNAs. These oligonucleotides bind to the complementary mRNA transcriptsand prevent translation. Absolute complementarily, although preferred,is not required. A sequence “complementary” to a portion of an RNA, asreferred to herein, is a sequence sufficiently complementary to be ableto hybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may be tested, or triplex formation may be assayed. The ability tohybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence, up to and including the AUGinitiation codon, are generally most efficient for inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have also been shown to be effective for inhibitingtranslation (Wagner, Nature, 372:333, 1984). Thus, oligonucleotidescomplementary to either the 5′ or 3′ non-translated, non-coding regionsof a JNK3 could be used in an antisense approach to inhibit translationof the endogenous human homolog of JNK3 mRNA. Oligonucleotidescomplementary to the 5′ untranslated region of the mRNA should includethe complement of the AUG start codon. Examples of candidate antisensesequences for the 5′ and 3′ regions are; 5′-AAG AAA TGG AGG CTC ATA AATACC ACA GCT-3′ (SEQ ID NO:17) and 5′-ATT GGA AGA AGA CCA AAG CAA GAG CAACTA-3′ (SEQ ID NO:18), respectively.

While antisense nucleotides complementary to the coding region of a JNK3gene could be used, those complementary to transcribed untranslatedregions are most preferred. Examples of this type of candidate sequenceare 5′-TAA GTA AGT AGT GCT GTA TGA ATA CAG ACA-3′(SEQ ID NO:19) and5′-TAC TGG CAA TAT ATT ACA GAT GGG TTT ATG-3′ (SEQ ID NO:20).

Antisense oligonucleotides complementary to mRNA coding regions are lessefficient inhibitors of translation, but could be used in accordancewith the invention. Whether designed to hybridize to the 5′, 3′, orcoding region of a JNK3 mRNA, antisense nucleic acids should be at leastsix nucleotides in length, and are preferably oligonucleotides rangingfrom 6 to about 50 nucleotides in length. In specific aspects, theoligonucleotide is at least 10 nucleotides, or at least 50 nucleotidesin length.

Regardless of the choice of target sequence, in vitro studies areusually performed first to assess the ability of an antisenseoligonucleotide to inhibit gene expression. In general, these studiesutilize controls that distinguish between antisense gene inhibition andnonspecific biological effects of oligonucleotides. In these studieslevels of the target RNA or protein are usually compared with that of aninternal control RNA or protein. Additionally, it is envisioned thatresults obtained using the antisense oligonucleotide are compared withthose obtained using a control oligonucleotide. It is preferred that thecontrol oligonucleotide is of approximately the same length as the testoligonucleotide, and that the nucleotide sequence of the oligonucleotidediffers from the antisense sequence no more than is necessary to preventspecific hybridization to the target sequence.

The oligonucleotides can be DNA or RNA, or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule or hybridization. The oligonucleotide may include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (as described, e.g., in Letsinger et al., Proc. Natl. Acad.Sci. USA 86:6553, 1989; Lemaitre et al., Proc. Natl. Acad. Sci. USA84:648, 1987; PCT Publication No. WO 88/09810) or the blood-brainbarrier (see, for example, PCT Publication No. WO 89/10134), orhybridization-triggered cleavage agents (see, for example, Krol et al.,BioTechniques 6:958, 1988), or intercalating agents (see, for example,Zon, Pharm. Res. 5:539, 1988). To this end, the oligonucleotide can beconjugated to another molecule, e.g., a peptide, hybridization-triggeredcross-linking agent, transport agent, or hybridization-triggeredcleavage agent.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethyl-aminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-theouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 2-(3-amino-3-N-2-carboxypropl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

The antisense oligonucleotide can also include at least one modifiedphosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal, or an analog of any of thesebackbones.

The antisense oligonucleotide can include an α-anomeric oligonucleotide.An α-anomeric oligonucleotide forms specific double-stranded hybridswith complementary RNA in which, contrary to the usual β-units, thestrands run parallel to each other (Gautier et al., Nucl. Acids. Res.15:6625, 1987). The oligonucleotide is a 2′-0-methylribonucleotide(Inoue et al., Nucl. Acids Res. 15:6131, 1987), or a chimeric RNA-DNAanalog (Inoue et al., FEBS Lett. 215:327, 1987).

Antisense oligonucleotides of the invention can be synthesized bystandard methods known in the art, e.g., by use of an automated DNAsynthesizer (such as are commercially available from Biosearch, AppliedBiosystems, etc.). As examples, phosphorothioate oligonucleotides can besynthesized by the method of Stein et al. (Nucl. Acids Res. 16:3209,1988), and methylphosphonate oligonucleotides can be prepared by use ofcontrolled pore glass polymer supports (Sarin et al., Proc. Natl. Acad.Sci. USA 85:7448, 1988).

The antisense molecules should be delivered to cells that express JNK3proteins in vivo. A number of methods have been developed for deliveringantisense DNA or RNA to cells; e.g., antisense molecules can be injecteddirectly into the tissue site, or modified antisense molecules, designedto target the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systemically.

However, it is often difficult to achieve intracellular concentrationsof the antisense molecule sufficient to suppress translation ofendogenous mRNAs. Therefore, an approach may be used in which arecombinant DNA construct comprises an antisense oligonucleotide placedunder the control of a strong pol III or pol II promoter. The use ofsuch a construct to transfect target cells in a patient will result inthe transcription of sufficient amounts of single stranded RNAs thatwill form complementary base pairs with the endogenous JNK3 transcriptsand thereby prevent translation of that mRNA. For example, a vector canbe introduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA.

Such vectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in mammalian cells.Expression of the sequence encoding the antisense RNA can be by anypromoter known in the art to act in mammalian, preferably human cells.Such promoters can be inducible or constitutive. Suitable promotersinclude, but are not limited to: the SV40 early promoter region(Bernoist et al., Nature 290:304, 1981); the promoter contained in the3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell22:787-797, 1988); the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci. USA 78:1441, 1981); and the regulatory sequencesof the metallothionein gene (Brinster et al., Nature 296:39, 1988).Constructs may also be contained on an artificial chromosome (e.g.,mammalian artificial chromosome; MAC; Harrington et al., Nature Genet.15:345-355, 1997).

The production of a JNK3 antisense nucleic acid molecule by any genetherapeutic approach described above results in a cellular level of JNK3protein that is less than the amount present in an untreated individual.

Ribozymes

Ribozyme molecules designed to catalytically cleave JNK3 mRNAs can alsobe used to prevent translation of these mRNAs and expression of JNK3mRNAs (see, e.g., PCT Publication WO 90/11364; Saraver et al., Science247:1222, 1990). While various ribozymes that cleave mRNA atsite-specific recognition sequences can be used to destroy specificmRNAs, the use of hammerhead ribozymes is preferred. Hammerheadribozymes cleave mRNAs at locations dictated by flanking regions thatform complementary base pairs with the target mRNA. The sole requirementis that the target mRNA have the following sequence of two bases:5′-UG-3′. The construction and production of hammerhead ribozymes iswell known in the art (Haseloff et al., Nature 334:585, 1988).Preferably, the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the JNK3 mRNA, i.e., to increaseefficiency and minimize the intracellular accumulation of non-functionalmRNA transcripts.

Examples of potential ribozyme sites in human JNK3 include 5′-UG-3′sites which correspond to the initiator methionine codon at, forexample, in human JNK3, about nucleotides 224-226, the codon for adownstream potential initiation site (nucleotides 338-340), andadditional codons in the coding region, including nucleotides 698-670;740-742; and 935-937.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”), such as the onethat occurs naturally in Tetrahymena Thermophila (known as the IVS orL-19 IVS RNA), and which has been extensively described by Cech and hiscollaborators (Zaug et al., Science 224:574, 1984; Zaug et al., Science,231:470, 1986; Zug et al., Nature 324:429, 1986; PCT Application No. WO88/04300; and Been et al., Cell 47:207, 1986). The Cech-type ribozymeshave an eight base-pair sequence that hybridizes to a target RNAsequence, whereafter cleavage of the target RNA takes place. Theinvention encompasses those Cech-type ribozymes that target eightbase-pair active site sequences present in JNK3 proteins.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, or targeting), andshould be delivered to cells which express a JNK3 gene in vivo, e.g.,the brain and spinal cord. A preferred method of delivery involves usinga DNA construct “encoding” the ribozyme under the control of a strongconstitutive pol III or pol II promoter, so that transfected cells willproduce sufficient quantities of the ribozyme to destroy endogenous JNK3mRNAs and inhibit translation. Because ribozymes, unlike antisensemolecules, are catalytic, a lower intracellular concentration isrequired for efficiency.

For any of the above approaches, the therapeutic JNK3 antisense orribozyme nucleic acid molecule construct is preferably applied directlyto the target area (e.g., the focal site of activity in a seizuredisorder, the hippocampus in Alzheimer's disease, the substantia nigrain patients with Parkinson's disease), but can also be applied to tissuein the vicinity of the target area or even to a blood vessel supplyingthe target area.

For gene therapy, antisense, or ribozyme JNK3 expression is directed byany suitable promoter (e.g., the human cytomegalovirus, simian virus 40,or metallothionein promoters), and its production is regulated by anydesired mammalian regulatory element. For example, if desired, enhancersthat direct preferential gene expression in cells under excitotoxicinduction can be used to direct antisense JNK3 expression in a patientwith a seizure disorder.

JNK3 antisense or ribozyme therapy is also accomplished by directadministration of the antisense JNK3 or ribozyme RNA to a target area.This mRNA can be produced and isolated by any standard technique, but ismost readily produced by in vitro transcription using an antisense JNK3DNA under the control of a high efficiency promoter (e.g., the T7promoter). Administration of antisense JNK3 RNA to target cells iscarried out by any of the methods for direct administration oftherapeutic compounds described herein.

Methods of Treating Disorders Involving JNK3 Expression or Activity

The invention also encompasses the treatment of disorders, especially inmammals, such as humans, in which JNK3 plays a damaging role. A numberof disorders or the nervous system involving excitotoxicity, such asseizure disorders (e.g., epilepsy), dementias such as neurodegenerativedisorders (e.g., Alzheimer's disease, Huntington disease),cerebrovascular disorders such as ischemia, motor neuron disease(including ALS), injuries caused by extreme heat or cold, trauma (e.g.,irradiation, spinal cord injury, pressure, and ionic imbalance),metabolic imbalance (e.g., hypoglycemia) and Parkinson's disease, can betreated by the methods described herein. Without limiting the inventionby committing to any particular theory, a substantial number ofneurologic disorders are attributable, at least in part, toexcitotoxicity which is mediated by the JNK3 pathway. Thus, inhibitorsof this pathway, identified as described above, are useful for treatmentof disorders involving excitotoxicity.

Therapy can be designed to reduce the level of endogenous JNK3 geneexpression, e.g., using antisense or ribozyme approaches to inhibit orprevent translation of a JNK3 mRNA; triple helix approaches to inhibittranscription of the gene; or targeted homologous recombination toinactivate or “knock out” a gene or its endogenous promoter. Theantisense, ribozyme, or DNA constructs described herein can beadministered directly to the site containing the target cells; e.g.,specific regions of the brain or the spinal cord. Antibodies orfragments of antibodies that recognize JNK3 or a JNK3 substrate, andthat have been modified to be expressed or otherwise enter the cell canalso be used therapeutically.

Effective Dose

Toxicity and therapeutic efficacy of the compounds of the invention,e.g., compounds that modulate JNK3 expression or activity, can bedetermined by standard pharmaceutical procedures, using either cells inculture or experimental animals to determine the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. Polypeptides or other compounds that exhibit largetherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue tominimize potential damage to uninfected cells and, thereby, reduce sideeffects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (that is, the concentrationof the test compound which achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Levels in plasma canbe measured, for example, by high performance liquid chromatography.

Formulations and Use

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents (forexample, pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (for example, lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(for example, magnesium stearate, talc or silica); disintegrants (forexample, potato starch or sodium starch glycolate); or wetting agents(for example, sodium lauryl sulphate). The tablets may be coated bymethods well known in the art. Liquid preparations for oraladministration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (for example, sorbitolsyrup, cellulose derivatives or hydrogenated edible fats); emulsifyingagents (for example, lecithin or acacia); non-aqueous vehicles (forexample, almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (for example, methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The preferred methods of administering the compositions of the inventionare by direct delivery of the compounds to the central nervous system,preferentially to the brain, especially near to or directly at the siteof the disorder, e.g., the hippocampus in the case of Alzheimer'sdisease, the substantia nigra in the case of Parkinson's disease, andthe focal site for seizure disorders. Accordingly, administration may beinto a ventricle, intrathecal, or intracerebral ventricular. Forexample, an Omaya reservoir-shunt with in-line filter can be surgicallyplaced into the cisternal space. A therapeutic compound in anappropriate excipient (e.g., phosphate-buffered saline) is instilledinto the shunt by injection on a prescribed basis.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin for use in an inhaleror insufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration byinjection, for example, by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, for example, containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The therapeutic compositions of the invention can also contain a carrieror excipient, many of which are known to skilled artisans. Excipientswhich can be used include buffers (for example, citrate buffer,phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids,urea, alcohols, ascorbic acid, phospholipids, proteins (for example,serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol,and glycerol. The nucleic acids, polypeptides, antibodies, or modulatorycompounds of the invention can be administered by any standard route ofadministration. For example, administration can be parenteral,intravenous, subcutaneous, intramuscular, intracranial, intraorbital,opthalmic, intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, transmucosal, or oral. The modulatory compound can beformulated in various ways, according to the corresponding route ofadministration. For example, liquid solutions can be made for ingestionor injection; gels or powders can be made for ingestion, inhalation, ortopical application. Methods for making such formulations are well knownand can be found in, for example, “Remington's Pharmaceutical Sciences.”It is expected that particularly useful routes of administration will benasal or by direct infusion into the central nervous system.

EXAMPLES Example 1 JNK3 Expression

A 351-bp sequence derived from the 5′ region of the mouse JNK3 cDNA(nucleotides 62-412) was labelled with [³²P] by random priming and usedas a probe to determine the tissue expression pattern of the JNK3 gene.Northern blot analysis was performed by standard methods on 2 mg samplesof poly (A)⁺ mRNA isolated from testis, kidney, skeletal muscle, liver,lung, spleen, brain, and heart. All Northern blots were probed with[³²P]-labelled β-actin as a control to ensure loading of similar amountsof RNA in each lane. A strong signal corresponding to a 2.7 kbtranscript, as well as a weak signal corresponding to a 7.0 kbtranscript, were detected in brain. A weak signal corresponding to a 2.7kb transcript was also detected in the heart. A signal corresponding toa 2.4 kb transcript was detected in the testis. JNK3 expression was notdetected in the other tissues examined.

In situ hybridization analysis has indicated that JNK3 is expressed inmany regions of the brain (Martin et al., supra). Total RNA (10 mg) wastherefore isolated from different regions of mouse brain (cerebellum,cerebral cortex, hippocampus, midbrain, thalamus, and brainstem) usingthe TRIzol reagent (Gibco-BRL), and analyzed by Northern blot using theJNK3 probe described above. A signal corresponding to a 2.7 kbtranscript was detected in all sections of the brain examined, and wasmost abundant in the hippocampus.

Example 2 Targeted Disruption of the JNK3 Gene

To generate JNK3-deficient mice, a targeting vector was designed toreplace an internal 4 kb Mscl-Spel JNK3 genomic fragment with a PGKneocassette. A map of the JNK3 gene, the JNK3 targeting vector, and thepredicted structure of the mutated JNK3 gene are shown diagrammaticallyin FIG. 6. Restriction enzyme sites are indicated (B, BamHI; Hp, HpaI;M, MscI; Nco, NcoI; R, EcoRI; Spe, SpeI). A 10-kb NotI-EcoRI (the NotIsite was vector-derived) JNK3 fragment was cloned from a λ FixII phagelibrary of a 129/Sv mouse strain (Stratagene Inc.). The targeting vectorwas constructed by inserting a 4.0 kb MscI fragment from the 5′ end ofthe JNK3 genomic fragment, a 1.6 kb PGK-neo cassette (Negishi et al.,Nature 376:435-438, 1995) and a 1.8-kb SpeI-NcoI fragment of the 3′ endof the JNK3 fragment into pBluescript KS vector (Stratagene Inc.) usingappropriate linkers. The targeting vector contains a 2.6-kb PGK tkcassette (Negishi et al., supra) flanking the 5′ end of the JNK3 genomicsequence for negative-selection of mutant ES cells (Mansour et al.,336:348-352, 1988). The region replaced in the JNK3 gene by thetargeting vector encompasses one and a half exons encoding amino acids211 to 267 of JNK3 (as shown in FIG. 5B). This region includes thetripeptide dual phosphorylation motif Thr-Pro-Tyr (TPY) that ischaracteristic of the JNK group and required for protein kinase activity(Dèrijard et al., supra). The two hatched boxes shown in the JNK3 locuscorrespond to subdomains VIII and IX (encoding amino acid residues189-267 in the JNK3 protein shown in FIG. 5B) of JNK3.

The targeting vector was linearized with NotI and electroporated intoW9.5 embryonic stem (ES) cells. Genomic DNA from transfectants resistantto G418 (200 mg/ml) (Gibco BRL) and gancyclovir (2 mM) (Syntex, PalaAlto, Calif.) were isolated and screened by Southern blot analysis.Southern blot analysis of 104 independent G418- andgancyclovir-resistant clones revealed three clones containing thedesired homologous recombination event (targeting frequency 2.9%).Chimeric mice were generated by injecting these ES cells into C57BL/6(B6) mouse blastocysts.

Southern blots of EcoRI-restricted DNA derived from the tails of thesechimeric mice were probed with the radiolabeled 351 bp JNK3 probe. EcoRIdigestion resulted in a 12 kb band corresponding to the wild-type(endogenous allele), and a 4.2 kb band corresponding to the mutant(disrupted allele).

Two clones mediated germline transmission of the disrupted JNK3 alleleinto the next generation of mice. Heterozygotes (+/−) were intercrossedto generate homozygous mutant mice (−/−) that were identified bySouthern blot analysis of genomic DNA. Total RNA isolated from mousebrain was examined by Northern blot analysis. The blot was probed with arandom-primed ³²P-labeled mouse JNK3 cDNA probe, then stripped andsequentially reprobed with mouse JNK1 and β-actin cDNA probes. The majorJNK3 transcript in brain is 2.7 kb, and the JNK1 transcripts in mousebrain are 2.3 and 4.4 kb. Blots hybridized with a JNK3 cDNA probedetected transcripts in wild-type (+/+), but not in homozygous knockout(−/−) mice.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis wasused to confirm that JNK3 transcripts were absent in the homozygousJNK3(−/−) brain. A JNK1 probe (447 bp) was amplified from mouse brainRNA by RT-PCR (Yang et al., supra) using the amplimers5′-GTGTGCAGCTTATGATGCTATTCTTGAA-3′ (SEQ ID NO:21) and 5′CGCGTCACCACATACGGAGTCATC-3′ (SEQ ID NO:22). RT-PCR detection (Yang etal., supra) of JNK3 mRNA in mouse tissues was performed by RT-PCR usingthe amplimers: 5′-CTGGAGGAGTTCCAAGATGTCTACT-3′ (SEQ ID NO:23) and5′-TGGAAAGAGCTTGGGGAAGGTGAG-3′ (SEQ ID NO:24) to yield a specific 537 bpDNA product. RNA isolated from mouse brain was amplified with primersspecific for HPRT as a control. These experiments confirmed the absenceof JNK3 transcripts in the homozygous JNK3(−/−) brain.

Protein kinase assays were performed to show that JNK3 (−/−) mouse brainwas deficient in JNK3 activity. In these experiments, JNK3 kinaseactivity in brain lysates was measured after immunodeletion of JNK1 andJNK2 by in-gel protein kinase assays using the substrate GST-cJun(Dèrijard et al., supra). When mouse hippocampal lysates (30 μg) fromwild type (+/+) and homozygous knockout (−/−) brains were assayed, the55 kD and 46 kD JNK3 isoforms were detected in wild type but notJNK3(−/−) mice, confirming that JNK3 (−/−) mouse brain was deficient inJNK3 kinase activity. Together, these data demonstrate that the targeteddisruption of the JNK3 gene resulted in a null allele.

The JNK3(−/−) mice were fertile and of normal size. Histological surveysof a variety of tissues revealed no apparent abnormality usinghematoxylin and eosin (H & E) staining of heart, lung, thymus, spleen,lymph nodes, liver, kidney, and skeletal muscle. JNK3(−/−) and wild-typemouse brains were examined by immunocytochemical analysis of a pyramidylneuronal marker (MAP-2), interneuronal markers calbindin andparvalbumin, an astrocyte marker (glial fibrillary acidic protein; GFAP;Hsu et al., J. Histochem. Cytochem. 29:577-580, 1981), and Nissl's stain(Hsu et al., supra). These studies revealed that JNK3(−/−) mice hadapparently normal development and structural organization of the brain.A comparable number of motor neurons were found in the facial nucleus inwild-type and JNK3(−/−) mice (2150-2300 neurons per nucleus at postnatalday 10, n=4). The neurons were identified by morphology and were countedby a double-blind assay of serial sections throughout the facial nucleiof wild-type and JNK3(−/−) mice. Thus, there is no apparentdevelopmental abnormality, including cell death, in JNK3(−/−) mice.

Example 3 JNK3 Deficient Mice are Resistant to KA-Induced Seizures

JNK3(−/−) mice and their wild-type littermates were injectedintraperitoneally (i.p.) with 30 mg/kg KA to induce seizures (Ben-Ari,supra). In wild-type mice, the administration of KA first induced“staring spells” with abnormal body posture, then progressed to headnodding (“wet-dog shakes”), fore-paw tremor, rearing, loss of posturalcontrol, and eventually, continuous convulsions. The seizure activitiestypically subsided one hour after injection. Wild-type and heterozygousmice developed motor symptoms of seizures, including rearing, at 30 to40 minutes post-injection. The JNK3(−/−) mice, in contrast, developedmuch milder symptoms, mainly consisting of “staring” spells andoccasional myoclonic tremors. At this dose, JNK3(−/−) mice did notdevelop grand mal seizures and recovered much faster than did wild-typeand heterozygous mice. JNK3(−/−) mice developed seizures of comparableseverity to wild-type mice only at higher dosages of KA (45 mg/kg,i.p.). However, at this high dose of KA, more than 60% of wild-type micedied from continuous tonic clonic convulsions, while all of theJNK3(−/−) mice survived. These results indicate that JNK3(−/−) mice wereresistant to the effect of the excitotoxin KA. Further, JNK3(−/−) micerecovered from the drug administration more rapidly than wild-type mice(FIG. 7). Seizure classifications as shown in FIGS. 7 and 8 are: 1,arrest of motion; 2, myoclonic jerks of the head and neck, with brieftwitching movements; 3, unilateral clonic activity; 4, bilateralforelimb tonic and clonic activity; and 5, generalized tonic-clonicactivity with loss of postural tone, often resulting in death.

Example 4 Resistance to Pentetrazole (PTZ)-Induced Seizures

Since the resistance to KA-induced seizures varied between littermates(+/+ and +/− are less resistant than −/− mice), the observeddifferential susceptibility cannot be attributed to a difference betweenmouse strains (Schauwecker and Steward, Proc. Nat. Acad. Sci. USA94:4103-4108, 1997). However, the resistance of JNK3 deficient mice toKA-induced seizures could be due to decreased drug penetration acrossthe blood-brain barrier or an increased GABA (gamma-aminobutyric acid)inhibitory postsynaptic potential (IPSP), or the ablation of a specificsignal transduction pathway mediated by the JNK3 protein kinase. Todistinguish between these possibilities, the response of JNK3(−/−) andwild-type mice to another epileptogenic agent, pentetrazole (PTZ)(Sigma), was examined. PTZ was selected due to its ability to induceseizures by blocking the GABA-IPSPs (Ben-Ari et al., Neurosci.6:1361-1391, 1981).

JNK3(−/−) mice and wild-type littermates developed seizures ofcomparable severity at all tested dosages of PTZ (30, 40, 50, 60 mg/kg,i.p.; FIG. 8). Moreover, unlike the slow progression of motor symptomsseen in the KA-induced seizures, PTZ induced abrupt general tonic-clonicseizures within five 10 minutes after injection, presumably reflectingthat its epileptogenic mechanism works solely through extracellularlyinhibition of the GABA-IPSP. Thus, the differential susceptibility to KAtoxicity in JNK3(−/−) mice can neither be explained as a consequence ofpoor drug delivery to the nervous system nor by potent GABA-IPSPs in theneural circuit. Furthermore, we examined the expression of thekainate-type glutamate receptor subunits GluR5-7 (Pharmingen cat. no.60006E) by immunocytochemistry using standard methods.

Pyramidal neurons in the hippocampal CA1 subfield were most prominentlylabeled by the Glu5-7 antibody. Both wild-type and JNK3(−/−) mice showedprominently labeled apical dendrites arising from lightly labeled somatain the CA1 subfield of the hippocampus, a pattern similar to the primatehippocampus (Good et al., Brain Research 624:347-353, 1993). In additionto kainate-type subunit GluR5-7, the expression pattern of the GluR1subunit that is essential to various glutamate receptor subtypes and theintracellular calcium-binding proteins parvalbumin and calbindin thatmay buffer the influx of extracellular calcium were alsoindistinguishable between JNK3(−/−) and wild-type mice. Together, theseresults indicate no apparent structural abnormality that might beresponsible for the resistance of JNK3(−/−) mice to KA-inducedexcitotoxicity.

Example 5 Attenuation of KA-Induced Phosphorylation of c-Jun

The systemic administration of KA in wild-type mice may induce astress-response pathway mediated by the JNK3 protein kinase. To explorethis possibility, the expression of the immediate-early genes c-fos andc-jun (Morgan et al., Annu. Rev. Neurosci. 14:421-451, 1991; Smeyne etal., Nature 363:166-169, 1993; Kasof et al., J. Neurosci. 15:4238-4249,1995) was examined to determine whether KA imposed an equivalent levelof noxious stimulation on wild-type and JNK3(−/−) mice. Total RNA wasextracted from the hippocampi of mice sacrificed before and at 0.5, 2,4, or 8 hours after KA injection (30 mg/kg, i.p.), and Northern blotswere probed with murine c-fos and c-jun probes. The c-Jun probe was a207 bp fragment corresponding to nucleotides 888-1094 (FIG. 9) of themurine c-Jun cDNA. The c-Fos probe was a 347 bp fragment of the murinec-Fos gene (exon 4; base pairs 2593-2939) (FIG. 10). Both JNK3(−/−) andwild type mice exhibited a comparable level of rapid induction of c-fosand c-jun transcripts, which gradually declined four hours afterinjection.

To further define this phenomenon, the distribution of KA-induced c-Fosand c-Jun immunoreactivity along the synaptic circuit of the hippocampuswas examined. In these experiments, homozygous mutant and controlwild-type mice were killed and fixed by transcardial perfusion of 4%paraformaldehyde at 2 or 6 hours after the injection of KA (30 mg/kg,i.p.). Brains from both groups were removed, post-fixed for one hour,and sectioned on a Vibratome (40 mm thick). Tissue sections wereprocessed by immunocytochemistry to detect the expression of c-Jun(Santa Cruz, cat#sc-45), c-Fos (Santa Cruz, cat#sc-52), andphospho-specific c-Jun (Ser-73) (New England Biolabs, #9164S). Sectionswere floated in a solution of the primary antibody (diluted 200× in PBS)and incubated overnight at room temperature. Secondary antibodyincubation, avidin-biotin conjugated peroxidase (Vectastain Elite ABCkit, Vector Lab.), and DAB (3,3′-diaminobenzidine, Sigma) reactions wereperformed using standard procedures (Hsu et al., supra). In the absenceof KA, there was no detectable c-Fos expression and only a fewc-Jun-positive cells within the dentate gyrus. Two hours after KAinjection (30 mg/kg, i.p.), there was a large increase in c-Fosimmunoreactivity throughout the hippocampal region that was the same inboth wild-type and JNK3(−/−) mice. Simultaneously, there was an increasein the number of c-Jun-positive cells in the dentate gyrus and the CA3region of the hippocampus in both wild type and JNK3(−/−) mice. By sixhours after KA injection, the expression of c-Jun extended to the CA1region in both wild-type and JNK3(−/−) mice. The induction of c-Fos andC-Jun is generally accepted as an indicator of neuronal activityfollowing noxious stimulation (Morgan et al., supra). The comparableinduction level, time-course, and distribution of c-Jun andc-Fos-labeled cells suggests that JNK3(−/−) and wild type mice weresubject to an equivalent level of noxious stress by systemicadministration of KA.

C-Jun is activated by phosphorylation of the NH₂-terminal activationdomain by JNK. The expression of phosphorylated c-Jun provides anothermeasure of whether JNK-like activity was present in JNK3(−/−) mice. Theexpression of phosphorylated c-Jun was examined using an antibody raisedagainst c-Jun phosphorylated at Ser-73, one of the sites phosphorylatedby JNK (Whitmarsh et al., supra; Dèrijard et al., supra; Kyriakis etal., supra). Prior to challenge with KA, no cells were labeled by theantibody in either wild type or JNK3(−/−) mice. By two hours after KAinjection, there was a high level of phosphorylated c-Jun in the dentategyrus and the CA3/CA4 region of the hippocampus in wild-type mice. Incontrast, only a trace amount of phosphorylated c-Jun was detected inthe JNK3(−/−) mice. Thus, there was either a decreased level or lesssustained phosphorylation of c-Jun in the JNK3(−/−) mice.

In addition, there was a dynamic change of the distribution ofphosphorylated c-Jun in the wild type mouse hippocampus. By six hoursafter KA injection, the expression of phosphorylated c-Jun subsided inthe dentate gyrus and progressed to a restricted area in the hippocampalCA3 region. Under higher magnification, it was apparent that theexpression of phosphorylated c-Jun surrounded a focus of celldestruction. In contrast, no labeling of phosphorylated c-Jun wasdetected in the JNK3(−/−) mice at the same time point. The hippocampalCA3 region is well documented as the most vulnerable structure to the KAexcitotoxicity, presumably due to both a high KA binding affinity(Berger et al., supra) and a potent excitatory synaptic connectionbetween CA3 pyramidal neurons (Westbrook et al., Brain Research273:97-109, 1983). These results indicate that JNK3 is required for thephosphorylation of c-Jun induced by KA.

Example 6 Attenuation of KA-Induced AP-1 Transcriptional Activity

Since the phosphorylation of c-Jun is an important initial event duringthe induction of AP-1 transcriptional activity (Whitemarsh et al.,supra; Yang et al., supra), whether the observed attenuation of c-Junphosphorylation would lead to decreased induction of AP-1transcriptional activity in JNK3(−/−) mice was examined. JNK(-1-) micewere crossed with transgenic AP-1 luciferase (AP1-luc) mice (Rincon etal., Embo. J. 13:4370-4381, 1994) and progeny back crossed. TheJNK3(−/−)/API-Luc(−/+) mice were used in experiments with JNK3(+/+) miceto compare the level of KA-induced AP-1 transcriptional activity in thepresence or absence of JNK3. The AP1-luc mice contain the fireflyluciferase gene under the control of four copies of a consensus AP-1binding site in the context of the minimal rat prolactin promoter. Ithas been established that the expression of luciferase in these mice isdue to the presence of the AP-1 regulatory element.

In the luciferase assay, mice were sacrificed at intervals afterinjection of KA (30 mg/kg, i.p.), and relative luciferase activitycompared with that detected in hippocampal lysates obtained from miceinjected with vehicle (saline). Mice were decapitated, brains weredissected, and brain tissues were immediately lysed in buffer containing25 mM Hepes pH 7.4, 1% TRITON®X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin (Promega, Madison, Wis.).Luciferase activity was measured as described in Rincon and Flavell(Embo. J. 13:4370-4381, 1994). The injection of KA (30 mg/kg, i.p.)caused a large induction of AP-1 transcriptional activity in thehippocampus of wild-type mice, as evidenced by the induction ofluciferase activity. Luciferase activity in wild type mice wasdetectable by six hours, gradually increased to the peak at three days,and persisted for at least seven days (FIG. 11). Control experimentsdemonstrated that the injection of vehicle (saline) did not causeinduction of luciferase activity in the AP1-luc mice.

The relative luciferase activity in the hippocampus and cerebellumprepared from wild-type (+/+) and JNK3 (−/−) mice was measured followingKA injection. The results are shown in FIG. 12. Each time pointrepresents the mean of three to five (+SEM) individual animals. Theinduction of luciferase activity was most prominent in the hippocampus,where a markedly greater induction of phosphorylation of c-Jun wasobserved, as compared to the cerebellum and the cerebral cortex. Theinduction of AP-1 activity was significantly reduced in the JNK3(−/−)mice with the AP1-luc transgene as compared to the wild type mice. At 15hours after injection of KA, there was approximately four-fold greaterAP-1 activity in the hippocampus of wild-type mice compared withJNK3(−/−) mice. At three days after injection, the AP-1 activity wasmore than six times higher in the hippocampus of wild-type compared withJNK3(−/−) mice. Together, these data demonstrate that the disruption ofthe JNK3 gene suppressed KA-induced phosphorylation of c-Jun and AP-1transcription activity in the hippocampus in vivo.

Example 7 Resistance to KA-induced Apoptosis

One unique feature of KA among other epileptogenic agents is its potencyin inducing neuronal cell death (Ben-Ari, supra; Schwob et al., supra).Since this property of cell destruction is paralleled by a sustainedlevel of AP-1 transcriptional activity, it has been suggested that AP-1mediates KA-induced-neuronal death (Kasof et al., supra; Schwarzschildet al., J. of Neurosci. 17:3455-3466, 1997). Wild-type and JNK3(−/−)mouse brains were therefore examined after treatment with KA todetermine whether the attenuation of AP-1 transcriptional activity inJNK3(−/−) mice altered the extent of neuronal damage (Ben-Ari, supra;Ben-Ari et al., supra; Schwob et al., Neurosci. 5:991-1014, 1980).

These experiments were performed as follows. Wild-type and JNK3(−/−)mice were killed and fixed by transcardial perfusion of 4%paraformaldehyde and 1.5% glutaraldehyde three days after the injectionof KA (30 mg/kg, i.p.). Semithin and thin sections of brain wereprepared using a Vibratome and embedded in Epon. Tissue blocks wereprepared using a microtome with a diamond tube for 1 μm-thick semithinsections examined by toluidine blue staining, and for ultrathin sectionsexamined by electron microscopy. Nissl's stain was used for initialexamination of damage to the hippocampus (Kluver et al., J. Neuropath.Exp. Neuro. 12:400-403, 1953). GFAP immunocytochemistry was also used toassess cell destruction in the hippocampus (Hsu et al., supra). Nissl'sstaining was also performed as described above. TUNEL assays, used toevaluate apoptosis, were performed using cryostat sections (50 μm) ofcerebral hemispheres that were cryoprotected with sucrose. The TUNELassay was modified from the terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling assay (Gavrieli et al., J. Cell.Biol. 119:493-501, 1992). Briefly, tissue sections, directly mounted ona salinated slide, were permeablized with 2% TRITON® X-100 (20 minutesat room temperature) and then incubated for nick end-labeling for 2hours at 37° C. using 0.32 U/μl TdT (Boehringer Mannheim, cat#220582)and 2 μM digoxigenin-11-dUTP (Boehringer Mannheim, cat#1573152) in afinal volume of 40 μl. The tissues were incubated with anti-digoxigeninantibody (Boehringer Mannheim, cat#1333062) diluted 500-fold, andprocessed for immunocytochemistry using standard procedures (Hsu et al.,supra).

The damage to the hippocampus caused by KA was initially examined byNissl's stain. The KA-induced cell loss caused either a breach ofstaining of the pyramidal neurons in the CA3 region or a diffuse sparsestaining throughout the CA1 subfield. To corroborate the celldestruction revealed by Nissl's stain, the TUNEL method was applied todetect apoptosis. Groups of small pyknotic nuclei and positivelyTUNEL-labeled cells were found in the hippocampal CA3 subfield devoid ofNissl's staining. Similarly, a high percentage of pyramidal neuronsshowing pyknotic nuclei and shattered apical dendrites and numerousstrongly TUNEL-labeled cells were located in the hippocampal CA1subfield which exhibited decreased Nissl's staining. Since the TUNELmethod and the pyknosis morphology only indicated the extent of celldamage at one time point of a dynamic process, immunostaining ofdamage-induced GFAP was also used as an independent assessment of theextent of cell destruction in the hippocampus. Consistent with thepatterns of Nissl's, toluidine, and TUNEL staining, an increased numberof strongly GFAP-labeled astrocytes was found either in the hippocampalCA3 or CA1 regions. Thus, a combination of Nissl's stain, GFAPimmunocytochemistry, TUNEL method, and toluidine stain of semithinsections was used to classify the KA-induced damage in the mousehippocampus.

A total of 17 wild-type and 18 JNK3(−/−) mice were examined. Results areshown in Table 1 (below). The table was compiled from two sets of data.First, wild-type (n=11) and JNK3(−/−) (n=10) mice were sacrificed on thefifth day after a single injection of KA (30 mg/kg, i.p.). Second,wild-type (n=6) and JNK3(−/−) (n=8) mice received an injection of KA (30mg/kg, i. p.) for five consecutive days and examined two days followingthe final injection. The severity of the hippocampal damage in wild-typemice was comparable in experiments using both protocols. The ratio of nocell loss/CA3 lesion/CA3+CA1 lesion was 2/7/2 in the single-injectionexperiments, and 2/2/2 in the multiple injection experiments.

TABLE 1 Kainate-induced neuronal damage (number of animals) JNK3genotype +/+ −/− No perceivable cell loss = 4 18 Selective CA3 cell loss9 0 Including CA1 cell loss 4 0

The hippocampal CA3 region was the most susceptible to KA-induced damagein the wild-type mice (9/17; 53%). Cell loss was indicated by decreasedcrystal violet staining in the CA3 region. Using the TUNEL method(Gavrieli et al., supra), which identifies DNA fragmentation in thedying cells, groups of labeled cells were found in the damaged region. Acluster of pyknotic nuclei was found in the CA3 region intoluidine-stained semithin sections. As a result of KA-induced damage,there was selective glial proliferation confined to the CA3 region, asindicated by the strong immunostaining of GFAP. In some wild-typeanimals, massive cell loss was observed throughout the entirehippocampal CA1 region (4/17; 24%). Similarly, damage to the CA1 regionwas revealed by decreased crystal violet staining, positivelyTUNEL-labeled cells, pyknotic nuclei, shattered apical dendrites ofpyramidal neurons, and both hypertrophy and proliferation ofGFAP-positive astrocytes.

In contrast, there was no apparent hippocampal damage in any of theJNK3(−/−) mice examined (n=18). The pattern of the Nissl's stain, TUNELassay, toluidine blue staining of semithin sections, and GFAPimmunostaining of the hippocampal region in the JNK3(−/−) mice wasindistinguishable from that of untreated wild-type mice. Moreover,although JNK2(−/−) mice developed seizures of comparable severity at thesublethal dose of 45 mg/kg KA (a dose that is lethal for more than 60%of wild-type mice due to continuous convulsions), cell damage wasnevertheless found in a much smaller percentage of animals (2/15, 13%;p<0.005 by chi-square analysis, d.f.=1).

Methods of assessing apoptosis (e.g., TUNEL assay) can be used toevaluate whether JNK3 modulator is affecting apoptosis.

Example 8 Electron Microscopic Analysis of Ultrastructural ChangesAssociated with KA-Induced Neuronal Damage

Cortical neurons in vitro undergo either apoptosis or necrosis dependingon the extracellular concentration of the glutamate analogN-methyl-D-aspartate (NMDA) (Bonfoco et al., Proc. Natl. Acad. Sci. USA92:7162-7166, 1995). The distinction between apoptosis versus necrosisin KA-induced neuronal damage is critical since necrosis is generallythought to represent a consequence of acute mechanical insult that isincompatible with an active cell death program involving de novo proteinsynthesis. The TUNEL results (supra) indicate the involvement ofapoptosis. To further examine whether the neuronal death in vivo due toKA induction was apoptotic or necrotic, electron microscopy was employedto investigate the ultrastructural changes in the degeneratedhippocampal neurons. The microscopic analysis suggested a series ofmorphological changes indicating neuronal damage in the wild-type mouseas a consequence of apoptosis. The initial event after KA injection (30mg/kg i.p.) appeared to be compaction and segregation of chromatin inpyramidal neurons into electron-dense masses that abutted on the innersurface of the nuclear envelope. In contrast, the nuclei of thehippocampal neurons in the JNK3(−/−) mice following KA injectioncontained homogeneous electron-lucent euchromatin. At later stages, inwild type mice, there was convolution of the nuclear outline andcondensation of the cytoplasm. The double-layered structure of thenuclear envelope in wild type mice remained largely intact in all ofthese morphological stages. Eventually the degenerated neuronsdisintegrated resulting in numerous membrane-bounded apoptotic bodies.These morphological features are all consistent with the hallmarks ofapoptosis (Kerr et al., Br. J. Cancer 26:239-257, 1972). Thus, itappeared that KA triggered a genetic program within the damaged neuronleading to apoptosis, which was abrogated in JNK3-deficient neurons.

These results suggest that KA-induced phosphorylation of theNH₂-terminal activation domain of c-Jun leads to increased AP-1transcriptional activity and neuronal apoptosis. Without limitation to aparticular theory, a proposed chain of molecular events caused by KAthat lead to neuronal apoptosis is shown in FIG. 13.

Although systemic administration of KA causes cell damage predominantlylocalized in the hippocampal CA3 area, the significance of JNK3 instress-induced neuronal apoptosis is not only restricted to this region.Several lines of evidence indicate that the particular vulnerability ofthe CA3 hippocampal neurons to KA is due to their unique cellular andsynaptic properties. First, the hippocampal CA3 and CA4 regions have thehighest density of KA-receptors (Berger et al., Neurosci. Lett.39:237-242, 1983). Second, the recurrent synaptic excitation isparticularly potent in the hippocampal CA3 region (Miles et al., J.Physiol. (London) 373:397-418, 1986). The recurrent excitation of theCA3 pyramidal neurons may sustain JNK3 signaling and therefore rapidlyinduce KA excitotoxicity. The observed progression of c-Junphosphorylation from the dentate gyrus to the CA3 region is reminiscentof the synaptic circuitry of the hippocampus. A diagram of thetrisynaptic connection within the hippocampal formation is shown in FIG.14. The first synaptic relay (1) is from the afferent perforant path(pp) onto the granule cell of the dentate gyrus (DG). The second relay(2) follows the mossy fiber (mf) from the dentate gyrus to the CA3hippocampal neurons. The third relay (3) is from the hippocampal CA3 tothe CA1 region along the Schaffer collaterals (Sch). There are recurrentsynaptic interactions of pyramidal neurons in the CA3 region.

Example 9 Assays for Detection of Inhibitors of JNK3 Protein KinaseActivity

Inhibitors of JNK3 can be identified in protein kinase assays. Theseassays can be performed using JNK3 purified from tissue (e.g., brain) orwith recombinant enzyme. The recombinant JNK3 can be isolated frombacteria, yeast, insect, or mammalian cells using standard procedures.Assays of endogenous (natural) JNK3 are known in the art and assays ofrecombinant JNK3 have been described previously (Gupta et al., EMBO J.15:2760-2770, 1996).

The protein kinase activity of JNK3 can be measured using ATP andprotein substrates for JNK3 in an in vitro assay. These substratesinclude, but are not limited to, the transcription factors ATF2 andElk-1 (Gupta et al., 1996, supra). The incorporation of phosphate intothe substrate can be measured by several methods. One example is tomeasure the incorporation of radioactive phosphate (e.g., ³²P) into thesubstrate. The incorporation into the substrate can be measuredfollowing removal of unincorporated radioactivity by precipitation withtrichloroacetic acid and recovery on phosphocellulose paper or bypolyacrylamide gel electrophoresis. The radioactivity can be monitoredby scintillation counting, phosphorimager analysis, or byautoradiography. In general, methods for automated high throughputscreens would not use radioactive materials. For this purpose a methodis used to detect the phosphorylated substrate without a radioactiveprobe. In one approach the electrophoretic mobility of the substrate isexamined. For example, ATF2 demonstrates a marked reduction inelectrophoretic mobility following phosphorylation by JNK on Thr-69 andThr-71 (Gupta et al., Science 267:389-393, 1995).

A second approach is to detect the phosphorylation of the substrateusing immunochemical methods (e.g. ELISA). Antibodies that bindspecifically to the phosphorylated substrates are prepared (monoclonaland polyclonal) and are commercially available (e.g., New EnglandBiolabs, Promega Corp., and Upstate Biotechnology Inc.). The extent ofsubstrate phosphorylation is then measured by standard ELISA assay usingsecondary antibodies coupled molecules suitable for tospectrophotometric or fluorometric detection using methods known in theart.

Molecules that inhibit JNK3 can be identified in a high throughputscreen. A molecule that is a preferred candidate to treat excitotoxicdisorders inhibits JNK3, but not other protein kinases, includingrelated MAP kinases. Candidate molecules once identified can beoptimized using combinatorial chemical methods or by the synthesis ofrelated molecules. These molecules represent candidate drugs that can betested for JNK3 therapy.

Example 10 Assays for Detection of Inhibitors of JNK3 Activation

The JNK protein kinases are activated by dual phosphorylation on Thr andTyr within protein kinase sub-domain VIII (Davis, Trends Biochem. Sci.19:470-473, 1994). These sites of activating phosphorylation areconserved in JNK3 (Gupta et al., 1996, supra). Molecules that inhibitthe activation of JNK3 by interfering with the phosphorylation of JNK3can be identified by measurement of JNK3 activation in the presence andabsence of candidate molecules. Cells expressing JNK3, e.g., neuronalcells, neuroendocrine cells, or cells that are engineered to expressrecombinant JNK3 (Gupta et al., 1996 supra), are exposed toenvironmental stress (e.g., depolarization, excitotoxic agents, UVradiation, heat, and anoxia) to activate JNK3. The state of JNK3activation can be assessed by several methods. For example, JNK3 can beisolated, washed free of the candidate inhibitor, and the activationstate of JNK3 monitored by protein kinase assay (supra). Alternatively,the activation of JNK3 can be probed using immunological methods usingantibodies that bind to the Thr and Tyr phosphorylated (activated) formof JNK3. Antibodies that bind specifically to the Thr and Tyrphosphorylated enzyme can be prepared (monoclonal and polyclonal) andare commercially available (e.g., from New England Biolabs and PromegaCorp.). The extent of substrate phosphorylation can then be measured bya standard ELISA assay using secondary antibodies coupled tospectrophotometric or fluorometric detection.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of identifying a compound that modulates JNK3 expression,the method comprising: incubating a cell that can express a JNK3 proteinwith a compound under conditions and for a time sufficient for the cellto express a JNK3 protein absent the compound; incubating a control cellunder the same conditions and for the same time absent the compound;measuring JNK3 expression in the cell in the presence of the compound;measuring JNK3 expression in the control cell; and comparing the amountof JNK3 expression in the presence and absence of the compound, whereina difference in the level of expression indicates that the compoundmodulates JNK3 expression.
 2. The method of claim 1, wherein thecompound decreases the expression of JNK3.
 3. A method of identifying acompound that modulates JNK3 activity, the method comprising: incubatinga cell that has JNK3 activity with a compound under conditions and for atime sufficient for the cell to express JNK3 activity absent thecompound; incubating a control cell under the same conditions and forthe same time absent the compound; measuring JNK3 activity in the cellin the presence of the compound; measuring JNK3 activity in the controlcell; and comparing the amount of JNK3 activity in the presence andabsence of the compound, wherein a difference in the level of activityindicates that the compound modulates JNK3 activity.
 4. The method ofclaim 3, wherein the compound decreases JNK3 activity.
 5. A method ofidentifying a compound that modulates the binding of a JNK3 polypeptideto a substrate, said method comprising comparing the amount of a JNK3polypeptide bound to a substrate in the presence and absence of aselected compound, wherein a difference in the amount of binding of aJNK3 polypeptide to a substrate indicates that said selected compoundmodulates the binding of a JNK3 polypeptide.
 6. The method of claim 5,wherein the binding of a JNK3 polypeptide to a substrate is decreased.7. A method for generating a totipotent mouse cell comprising at leastone inactivated JNK3 gene, the method comprising: a. providing aplurality of totipotent mouse cells; b. introducing into the cells a DNAconstruct comprising a disrupted mouse JNK3 gene, wherein the JNK3 geneis disrupted by insertion of a nucleotide sequence into the gene thatprevents expression of functional JNK3; c. incubating the cells suchthat homologous recombination occurs between the chromosomal sequenceencoding JNK3 and the introduced DNA construct; and d. identifying atotipotent mouse cell comprising at least one inactivated JNK gene.
 8. Amethod for generating a mouse homozygous for an inactivated JNK3 genecomprising: a. providing a totipotent mouse cell comprising at least oneinactivated JNK3 gene; b. inserting the cell into a mouse embryo andimplanting the embryo into a female mouse; c. permitting the embryo todevelop into a neonatal mouse; d. permitting the neonatal mouse to reachsexual maturity; and e. mating two sexually mature mice of step d. toobtain a mouse homozygous for the inactivated JNK3 gene(−/−), whereinthe homozygous JNK3(−/−) mouse is resistant to excitotoxic damage.
 9. Amethod of treating a patient having or at risk for a disorder involvingexcitotoxicity, the method comprising administering to the patient atherapeutically effective amount of a compound that inhibits JNK3expression.
 10. The method of claim 9, wherein the compound is anantisense nucleic acid molecule.
 11. The method of claim 9, wherein thedisorder is selected from the group consisting of Alzheimer's disease,Huntington disease, ischemia, amyotrophic lateral sclerosis, trauma,motorneuron disease, Parkinson's disease, or epilepsy.
 12. A transgenicnon-human mammal having a transgene disrupting expression of a JNK3gene, the transgene being chromosomally integrated into germ cells ofthe mammal.
 13. The mammal of claim 12, wherein the mammal is a mouse.14. The mammal of claim 12, wherein the germ cells are homozygous forthe transgene.
 15. The mammal of claim 12, wherein the disruptionresults in a null mutation.
 16. A cell line descended from a cell of themammal of claim
 12. 17. A DNA construct comprising a disrupted mouseJNK3 gene, including an insertion of a nucleotide sequence into the genethat prevents or modifies the expression of functional JNK3.